Stem cells, such as embryonic stem cells and somatic stem cells, are capable of differentiating into various types of cells in vitro. As a method of differentiating stem cells in vitro, there is utilized a method involving subjecting stem cells to floating culture to form pseudo-embryos, called embryoid bodies, or a method involving coculturing cells, such as stromal cells, that support differentiation and proliferation, with stem cells. Of those, floating culture is the most common method of differentiating stem cells in vitro. For example, when mouse embryonic stem cells are subjected to floating culture in a culture vessel, for example, a petri dish, without leukemia inhibitory factor (LIF), so as not to adhere to the vessel, cell aggregates are formed. The cell aggregates formed by floating culture are called embryoid bodies (EB). It is known that the resultant cell aggregates differentiate into various types of cells thereafter.
The embryoid body (EB) has a ball-like structure formed of a bilayer of cells. The outer layer corresponds to visceral endoderm, and the inner layer corresponds to embryonic ectoderm. The two endoderms are separated by a basement membrane. The structure of the embryoid body is quite similar to that of a cylindrical embryo, which is a day 6 mouse embryo. As far as this similarity is concerned, the structure resembles the normal stage of embryogenesis. In embryoid bodies, mesoderm is also induced, and cardiomyocytes, blood cells, and even primitive vascular networks are developed. In addition, when plated on a culture petri dish and cultured further, the embryoid bodies differentiate into various types of cells, including, for example, neurons, keratinocytes, chondrocytes, and adipocytes. It has recently been confirmed that the cells that differentiate via formation of embryoid bodies are differentiated not only into somatic cells, but also into a germ cell lineage. For utilizing pluripotency of stem cells, it is suitable that the embryoid bodies be formed.
In general, as a technology for forming cell aggregates, there are known a hanging drop method involving culturing cells in hanging drops, and a rotary culture method or a centrifugal method disclosed in Non Patent Literature 1. However, in each of those methods, setting of culture conditions is complicated.
In Patent Literature 1, as a vessel for forming cell aggregates, there is disclosed a culture vessel formed of polyhydroxylethyl methacrylate, an ethylene-vinyl alcohol copolymer, or the like.
In addition, in Patent Literature 2, there is disclosed a method involving subjecting ES cells, which are one type of stem cells, to floating culture to form embryoid bodies. In the method of forming embryoid bodies of Patent Literature 2, a vessel for embryoid body formation coated with a polymer having a phosphorylcholine-like group is used. In Patent Literature 2, there is disclosed a method of coating the vessel for embryoid body formation with the polymer, but detailed investigations are not conducted.